In current maize, or corn, production in the United States, the vast majority of the seed maize utilized by commercial farmers are singlecross, F1 hybrid varieties. The current commercial techniques for producing hybrid maize seed require that predictable cross-breedings be achieved between specifically selected male and female parent plants. Thus plants of the designated male and female lines are planted together in a common field so that pollen from the male parent plants can travel and pollinate the female parent plants.
This procedure is facilitated by the hermaphroditic character of maize plants. Each plant has separate male and female inflorescenses. Thus, in order to ensure that a proper cross is made between the desired male parent plant and the desired female parent plant, it is necessary to ensure that pollen from the female parent plant does not self-pollinate that same plant or pollinate a sibling female parent plant. In order to ensure that such undesirable self-pollination or sibling pollination does not occur, the common practice in the industry is to remove physically the male infloresena from the designated female parent plants by detasselling the female parent plants by hand. While mechanical devices are presently available in the art to detassel the female parent plants, because of the variability in size of maize plants in any given field and the necessity for not cutting too much of the maize plant away, mechanical processing is not efficient, and thus the detasselling procedure is conventionally done by hand, sometimes in combination with a mechanical device. This process is a very labor-intensive activity and is concentrated in its time period, normally a time period of four to six weeks in June, July or August, in the northern hemisphere, because the activity must be performed at closely spaced intervals during the flowering period of the maize inbred used as a female parent. This detasselling operation is both a difficult logistical operation, because of the need to acquire large amounts of short-term labor, and is an expensive process because of its labor intensiveness.
Accordingly, much effort has been spent over time to develop maize plants which are male-sterile. The term male-sterile generally designates a plant wherein the male inflorescenses organs on the mature plant produce no viable pollen, but the plant still has complete female reproductive capability. The use of male-sterile maize plants in a hybrid production system avoids the need for detasselling, since the only pollen available for the designated female parent plants, which are male-sterile, is the pollen produced by the designated male parent plant. In this way predictable crosses can be made so that hybrid progeny suitable for field use can be created. Unfortunately, the use of male sterile maize plants has previously had several inherent disadvantages.
There are two categories of presently known and used systems for maintaining male-sterile stocks of maize plants. One system relies on a so-called cytoplasmic, or non-nuclear, male-sterile trait, and the other system relies on genic, or nuclear, trait inheritance to maintain male sterility.
The cytoplasmic male sterility system relies on genes not contained in the nucleus of cells, hence the name. This system is more properly termed cytoplasmic-nuclear since it depends on both a cytoplasmic gene and the absence of a nuclear restorer genes for male sterility. Since cytoplasmic genetic material is normally transmitted solely from the female parent plant in maize, and is only very rarely, if ever, passed through pollen, the use of a cytoplasmic male-sterile trait in a female parent line allows pollen to be donated by a male-fertile parent while the resulting progeny plants are reliably male-sterile because of the cytoplasmic gene contribution of the female parent. One system disclosed for use of cytoplasmic male sterility to produce commercial hybrid maize is disclosed in U.S. Pat. No. 2,753,663.
For a time the United States hybrid seed industry utilized cytoplasmic male sterile maize lines for the production of hybrid maize seed. The most popular type of cytoplasmic male sterility was referred to as the Texas-Sterile or T-Sterile cytoplasm. This cytoplasmic sterility was used widely in producing several types and varieties of hybrid seeds for sale until 1970 when an epiphytotic of a race of T-type Helminthosporium maydis occurred causing a form of southern leaf blight in most of the then existing male-sterile plants and hybrids produced from them. This event convinced many maize breeders that cytoplasmic male sterility was an inherently inappropriate mechanism for achieving male-sterile plants since the differences between normal cytoplasm and that carrying male sterility seems inherently to affect not only pollen fertility but also disease susceptability as well. In addition, the heavy damage caused by this epiphytotic event has created a widespread reluctance to use cytoplasmic male-sterile lines because of consumer fears about reoccurrence of an epiphytotic in other cytoplasmic male-sterile lines. To date, two other cytoplasmic male sterility lines have been identified. Referred to as the C and S types, these types have inherent problems of stability and sterilization of inbred lines in addition to the normal consumer and breeder reluctance to use a cytoplasmic male-sterile system.
The other basis for male-sterility in maize plants is genic male sterility in which the nuclear genes of the maize plant cause male-sterility. Much work has been done on identification of male-sterile genes in maize, and, to date, at least nineteen different nuclear gene mutations are known which can produce male sterility. See the list of male-sterile genes, for example, in Column 15 of U.S. Pat. No. 3,861,079. In every presently known heritable trait which produces male sterility, the sterility is determined by a single gene, and the allele for male sterility is recessive. The possibility of using genic male-sterile lines has long been available to producers of hybrid seed but has not proved very practical to use.
The problem with the use of conventional genic male sterile lines is that it is inherently impossible to maintain an inbred stock which is homozygous for the recessive allele giving rise to male-sterility. The reason for this is simply that plants of the line are incapable of producing the pollen necessary to self-pollinate or pollinate siblings homozygous for the recessive allele. It is, of course, possible to cross-pollinate male sterile plants homozygous for the male-sterile recessive allele with male-fertile plants which are heterozygous for the male-sterile gene, i.e. having in their genic pair one male-fertile allele and one male-sterile allele (Ms/ms). The progeny from this cross-breeding would be approximatey 50% male-sterile and approximately 50% male-fertile. Thus if it was intended to use plants from a simple genic male-sterile line as female parents in a cross-fertilization scheme, the best that could be expected is that 50% of the progeny plants intended for use in hybrid seed production would be male-sterile. Therefore a detasselling operation would be necessary, in any event, to detassel the other 50% of the plants. Since detasselling is thus necessary anyway in a field utilizing this procedure, there is little commercial advantage to using this process, and it is not widely used at present.
One other approach to create genic male-sterile plants for use in hybrid seed production has been documented. A technique developed by Patterson utilizes reciprocal translocations and various forms of chromosome deficiencies and duplications to produce male sterile stocks. This procedure is described in detail in the disclosures of U.S. Pat. Nos. 3,710,511 and 3,861,079. That procedure need not be described in detail herein except to suggest that the approach that it uses to create genic male-sterile plants differs completely from the approach suggested herein, although the objective of both approaches is to produce genic, as opposed to cytoplasmic, male-sterile plants for hybrid seed production.
The system of the present invention makes use of one example of a class of genetic elements known as transposable elements. Transposable elements are genetic elements which can spontaneously relocate themselves from one locus to another on a chromosome or to anywhere on any other chromosome located in the plant genome. Transposable elements were first identified in maize, in the pioneering work of Dr. Barbara McClintock. Several systems of transposable elements have been identified by Dr. McClintock initially, and others subsequently. Transposable elements have now been identified in other species, including bacteria and animals, but are best characterized in maize.
Among the systems of transposable elements identified by McClintock, the system of particular interest to us is the Suppressor-mutator (Spm) system (McClintock, 1955). The same system was independently isolated by Peterson (1953) who referred to the components as Enhancer (En) and Inhibitor (I). Spm is a transposition-competent (autonomous) element which encodes the information enabling its excision from one location in the genome and reintegration at another location. It affects the expression of any locus in which it inserts. A second component may also be present, constituting a two element system. This second component has been shown to be a defective Spm in which a portion of the DNA sequence of Spm has been deleted (Pereira et al., 1985) and which has concomitantly lost the ability to catalyze its own transposition, but it can be induced to transpose when an Spm is present in the genome. This transpositiondefective derivative of Spm has been variously referred to as the receptor of Spm, Rs, and I. We will refer to this second component as a defective Spm (dSpm), since its origin is now understood in molecular terms, and also as a receptor factor (Rs), which defines its functional role.
These defective Spms can transpose from one locus and reintegrate in a second locus when an active Spm is present in the genome. In the absence of Spm, a dSpm is stably integrated in a locus where it may or may not affect the phenotype of the plant homozygous for that locus (McClintock, 1965). When an Spm is present in the genome, it suppresses all activity of any locus in which a dSpm has inserted thereby producing a mutant (null) phenotype until the dSpm responds to a trans-acting signal from Spm by being excised from the locus. If this excision event restores the normal organization of the locus in a cell, that cell and its clonal descendants will have a nonmutant (normal or wild type) phenotype. It is the possibility that a dSpm can integrate into a locus in such a manner that a nonmutant phenotype is produced in the absence of an active Spm but in the presence of an active Spm, gene activity is suppressed and a mutant phenotype is produced, that is of particular importance to this process of hybrid seed production. The Spm transposable element system is being used as a switch to turn off gene activity and produce male-sterile plants when desired for the purpose of hybrid seed production.
In this connection, it is desirable although probably not necessary to produce and utilize Spm's that have attenuated or null mutator activity in order to eliminate the possibility that an Spm-catalyzed excision of a dSpm integrated into an Ms allele (and producing a male-sterile phenotype in the presence of an active Spm) can occur sufficiently early in plant development to produce male-fertile sectors in the tassel. Thus, we desire Spm elements which are themselves partially defective having lost mutator activity but retaining Suppressor activity. McClintock has reported such partially defective Spms.